Multiplexed VeraCode bead-based serological immunoassay for colorectal cancer

J Immunol Methods. 2013 Dec 31;400-401:58-69. doi: 10.1016/j.jim.2013.09.013. Epub 2013 Oct 24.


Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US and Western world. Despite increased screening and advances in treatment, the mortality rate (ca. 50,000/year) and high national health-care burden for CRC are likely to remain high unless an effective non-invasive screening test for CRC is instituted for a large segment of the population. Blood-based protein biomarkers hold great promise for early disease diagnosis and personalized medicine; yet robust and reproducible multiplexing platforms and methodologies have lagged behind their genomic counterparts. Here, we report the development of a novel, multiplexed, hybrid immunoassay for CRC that is formatted on barcoded VeraCode™ micro-beads, which have until now only been used for genomic assays. The method combines a sandwich immunoassay format for detection of serum protein biomarkers with an antigen assay for autoantibody detection. The serum protein biomarkers CEA and GDF15 as well as autoantibodies to the p53 tumor associated antigen (TAA) were used to exemplify the method. This multiplex biomarker panel was configured to run on Illumina's holographically barcoded VeraCode™ micro-bead platform, which is capable of measuring hundreds of analytes simultaneously in a single well from small volumes of blood (<50 μL) using a 96-well industry standard microtiter plate. This novel use of the VeraCode™ micro-bead platform translates into a potentially low volume, high throughput, multiplexed assay for CRC, for the purposes of biomarker validation, as well as patient screening, diagnostics and prognostics. In an evaluation of a 186 patient sera training set (CRC and normal), we obtained a diagnostic sensitivity of 54% and a specificity of 98%. We anticipate that by expanding and refining the biomarkers in this initial panel, and performing more extensive clinical validations, such an assay could ultimately provide a basis for CRC population screening to complement the more invasive, expensive and low throughput (but highly sensitive and specific) colonoscopy.

Keywords: Autoantibodies; Biomarker; CEA; CRC; Colorectal cancer; GDF15; Multiplex immunoassay; TAAs; Tumor-associated antigens; carcinoembryonic antigen; colorectal cancer; growth differentiation factor 15; tumor associated antigens.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Autoantibodies / blood
  • Biomarkers, Tumor / blood*
  • Carcinoembryonic Antigen / blood*
  • Colorectal Neoplasms / diagnosis*
  • Early Detection of Cancer
  • Enzyme-Linked Immunosorbent Assay / methods
  • Growth Differentiation Factor 15 / blood*
  • High-Throughput Screening Assays
  • Humans
  • Immunologic Tests / methods*
  • Microspheres
  • Miniaturization
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tumor Suppressor Protein p53 / immunology


  • Autoantibodies
  • Biomarkers, Tumor
  • Carcinoembryonic Antigen
  • GDF15 protein, human
  • Growth Differentiation Factor 15
  • Tumor Suppressor Protein p53